The purpose of this project is to seek experimental evidence to support the recent hypothesis that enzymes obtain the energy needed for catalysis by forming very strong, low-barrier hydrogen bonds in the transition state, or in otherwise unstable intermediates, while having only weak hydrogen bonds in the ground state. Low-barrier hydrogen bonds have very low field proton NMR chemical shifts, and low deuterium fractionation factors. Thus the low field proton NMR signals of chymotrypsin and trypsin that have been assigned to the proton between aspartate and histidine in the catalytic triad will be examined by proton and deuterium NMR to determine the chemical shift, the isotope effect on the chemical shift, and (by integrating the signal in an H2O-D2O mixture) the fractionation factor. This will be done under conditions where the histidine is protonated (low pH, or in the presence of tetrahedral complexes with specific inhibitors). Low-barrier hydrogen bonds will be characterized in model compounds that resemble the asparate-histidine structure in serine proteases, such as hydrogen cis-urocanate and hydrogen 2-(4-imidazole)-2-methylpropionate, using NMR and X-ray methods. The effect of internal low-barrier hydrogen bonds on the chemical reactivity of these compounds will be determined. A number of other compounds that potentially could form internal low- barrier hydrogen bonds in appropriate solvents will also be studied. Proton NMR will be used to look for low-barrier hydrogen bonds that stabilize "carbanion" intermediates in the reactions catalyzed by enolase and fumarase, using alternate substrates or inhibitors where previous kinetic studies indicate that the intermediate may be present in appreciable concentration at equilibrium. The signals will be characterized as outlined above.